The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rossi, B.
Right arrow Articles by Gauthier, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rossi, B.
Right arrow Articles by Gauthier, L.
The Journal of Immunology, 2006, 177: 796-803.
Copyright © 2006 by The American Association of Immunologists

Clustering of Pre-B Cell Integrins Induces Galectin-1-Dependent Pre-B Cell Receptor Relocalization and Activation1

Benjamin Rossi, Marion Espeli, Claudine Schiff2 and Laurent Gauthier3

Centre d’Immunologie de Marseille-Luminy (CIML), Université de la Méditerranée, Case 906, Marseille, France; Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 631, Marseille, France; and Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 6102, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interactions between B cell progenitors and bone marrow stromal cells are essential for normal B cell differentiation. We have previously shown that an immune developmental synapse is formed between human pre-B and stromal cells in vitro, leading to the initiation of signal transduction from the pre-BCR. This process relies on the direct interaction between the pre-BCR and the stromal cell-derived galectin-1 (GAL1) and is dependent on GAL1 anchoring to cell surface glycosylated counterreceptors, present on stromal and pre-B cells. In this study, we identify {alpha}4beta1 (VLA-4), {alpha}5beta1 (VLA-5), and {alpha}4beta7 integrins as major GAL1-glycosylated counterreceptors involved in synapse formation. Pre-B cell integrins and their stromal cell ligands (ADAM15/fibronectin), together with the pre-BCR and GAL1, form a homogeneous lattice at the contact area between pre-B and stromal cells. Moreover, integrin and pre-BCR relocalizations into the synapse are synchronized and require actin polymerization. Finally, cross-linking of pre-B cell integrins in the presence of GAL1 is sufficient for driving pre-BCR recruitment into the synapse, leading to the initiation of pre-BCR signaling. These results suggest that during pre-B/stromal cell synapse formation, relocalization of pre-B cell integrins mediated by their stromal cell ligands drives pre-BCR clustering and activation, in a GAL1-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cells differentiate in the bone marrow (BM)4 and can be divided into successive subsets based on the expression of particular cell surface markers and on the sequential rearrangement of the genes coding for the Ig chains (1, 2). Completion of V to DJ rearrangements at the pre-BI stage leads to the production of the Ig µ (Igµ) H chain. Some of these Igµ chains associate with the surrogate L chain (SLC), composed of {lambda}-like (or {lambda}5) and VpreB, and with the signaling molecules CD79a and CD79b, to form the pre-BCR. Cells expressing the pre-BCR differentiate into the pre-BII stage and start to proliferate. Then, the SLC expression, and consequently that of the pre-BCR, is down-regulated leading to the arrest of proliferation and to the initiation of IgL gene rearrangements. Finally, cells start to express the BCR, composed of the Igµ and IgL chains associated with CD79a and CD79b, and become immature B cells which are selected on the basis of their BCR specificity and exported to the peripheral blood (3).

Precursor B cells develop in close association with a highly organized three-dimensional BM microenvironment. Adhesion molecules, including CD44, selectins, and integrins, control the interaction between B cell progenitors, the extracellular matrix (ECM) components, and BM stromal cells. Integrins are heterodimeric transmembrane molecules consisting of an {alpha} and beta subunit that mediate adhesion, migration, survival, and differentiation of the cells (4). They bind ECM components such as fibronectin and laminin, but also cellular receptors such as VCAM-1. The {alpha}4 subunit, that forms heterodimers with either beta1 or beta7 subunits, is crucial for normal hemopoiesis (5). In addition to providing physical support, BM stromal cells secrete soluble factors (IL-7, stromal cell-derived factor-1 (CXCL12), stem cell factor, Flt-3L), which regulate precursor B cell growth, maturation, and survival (6, 7, 8).

Pre-BCR expression represents a crucial step in B cell differentiation. The pre-BCR is implicated in pre-BII cell differentiation and proliferation (9, 10), in allelic exclusion of the IgH locus (11) and in the selection of the Igµ chain repertoire (12, 13). However, the mechanisms triggering the activation of the pre-BCR are not completely resolved (14, 15, 16). It has been shown that a fraction of the pre-BCR is associated with raft structures leading to its constitutive activation, and that pre-BCR engagement enhances this association, resulting in calcium flux and changes in protein tyrosine phosphorylation (17). In support of the existence of external pre-BCR ligands, it was recently demonstrated that the murine pre-BCR could specifically interact with stromal cell-associated heparan sulfates (15). Moreover, we reported the identification of the S-type lectin Galectin-1 (GAL1), expressed by stromal cells, as a human pre-BCR ligand (14). We observed that GAL1 binds to the NH2-terminal {lambda}-like chain of the SLC by protein-protein interactions and to glycosylated counterreceptors present at the cell surface of stromal and pre-B cells. GAL1 binding to glycosylated counterreceptors leads to pre-BCR clustering into the pre-B/stromal cell synapse and initiates intracellular tyrosine kinase activity and signal transduction from the pre-BCR.

In this study, we identify integrin family members as major GAL1-glycosylated counterreceptors present on both stromal and pre-B cells. Integrins and integrin ligands are polarized together with GAL1 and the pre-BCR into the pre-B/stromal cell synapse. We also demonstrate that pre-B cell integrin capping is sufficient to promote relocalization and activation of the pre-BCR, in the presence of GAL1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines, Abs, and reagents

The human Nalm6 pre-B cell, the human C8, and the murine MS5.1 stromal cell lines have been described previously (14). The anti-human VpreB 4G7 mAb has been described previously (18). The hamster anti-mouse CD3{epsilon} and the mouse anti-phosphotyrosine (4G10) mAbs were a gift from H. T. He (CIML, Marseille, France). The rabbit anti-human GAL1 antiserum (AS) was generated by Eurogentec using the human recombinant (hr) GAL1 protein (see below). The mouse HUTS anti-human beta1 mAb and the rat anti-L1 (19) mAbs were a gift from J. Marvaldi (Faculté de Pharmacie, Marseille, France) and G. Rougon (IBDM, Marseille, France), respectively. All other commercial Abs or reagents used in this study are listed in Table I.


View this table:
[in this window]
[in a new window]
  		Table I. Abs and reagents useda

 
Production and purification of recombinant proteins

The hrGAL1 and hrGAL1-M2-His proteins were obtained after C8 cell line cDNA amplification using coding 5'-ACCATGGCTTGTGGTCTGGTCG-3' and either noncoding 5'-AAAGCTTTTAGTCAAAGGCCACACATTTGATC-3' or 5'-AAGATCTCTTGTCATCGTCGTCCTTGTAGTCAAAGGCCACACATTTGATC-3' oligonucleotides, respectively, and cloning into the pQE-60 expression vector (Qiagen). Recombinant proteins were expressed in BL21-RP strain (Stratagene) and purified on {alpha}-lactose-agarose column (Sigma-Aldrich) followed by Superdex 75 size exclusion chromatography (AKTA system; Pharmacia Biosensor). The hrGAL1 and hrGAL1-M2-His proteins appear at the expected sizes, i.e., 14 and 15.5 kDa, respectively, on SDS-polyacrylamide gel (data not shown).

Biochemical analysis

Large scale preparative biochemistry was performed using the MS5.1 cell line as described (14). Nickel Sepharose beads were loaded with 4 mg of hrGAL1-M2-His. Urea-eluted fractions were analyzed on a SDS 7.5–17.5% gradient PAGE and fractions containing specifically eluted proteins were separated on a preparative SDS 7.5–17.5% PAGE and analyzed by mass spectrometry, as already described (14).

MS5.1 cells (10 x 106) were washed in ice-cold PBS, 0.2% BSA, 0.05% NaN3 (PBA buffer) twice before lysis in 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Igepal, 1 mM 2-ME, and protease inhibitor mixture lysis buffer (Sigma Aldrich). Proteins were immunoprecipitated for 2 h at 4°C using the hamster mAb anti-beta1 integrin coated on protein G-Sepharose beads, separated on a SDS 7.5–17.5% gradient PAGE, transferred to a nitrocellulose membrane, and revealed by Western blotting (14).

Nalm6 cells (20 x 106) were washed twice in ice-cold PBA buffer and incubated 30 min with 10 µg/ml hrGAL1 in ice-cold PBA buffer, 1 mM 2-ME. After washes, cells were lysed in the lysis buffer with either 0.2 M lactose or 0.2 M maltose. Proteins were immunoprecipitated for 3 h at 4°C using appropriate Abs coated on protein G-Sepharose beads. Immunoprecipitated proteins were separated and revealed as above.

Latex beads (25 x 106) were loaded with 10 µg/ml anti-beta1 and -CD9 mAbs, saturated with 5% BSA and washed in PBS. Nalm6 cells (50 x 106) were preincubated for 15 min with hrGAL1 (10 µg/ml) on ice before incubation with the beads for 30 min at 37°C. Nalm6 conjugates were then lysed for 15 min at 4°C with the lysis buffer complemented with 1 mM Na3VO4, 10 mM NaF, and 10 mM sodium pyrophosphate. Proteins were then immunoprecipitated for 3 h at 4°C using anti-CD79a mAb coated on protein G-Sepharose beads. Immunoprecipitated proteins were separated and revealed as previously described (14), using sequentially, mouse anti-phosphotyrosine mAb (4G10), mouse anti-CD79a mAb, and rabbit anti-Lyn Abs.

Far Western blotting

Lysates from the MS5.1, C8, and Nalm6 cell lines (160 µg of protein/lane) were run on a 7.5–17.5% SDS-PAGE and transferred to a nitrocellulose membrane saturated 20 min in PBS, 0.05% Tween 20, 5% nonfat dry milk, and then incubated for 1 h with or without hrGAL1 (2.5 µg/ml). The hrGAL1 protein was detected using the rabbit anti-GAL1 AS (1/5000 dilution), and revealed by HRP-coupled protein A.

Confocal microscopy

Confocal microscopy was performed as described (14). For actin polymerization experiments, Nalm6 cells (106/ml) were incubated with latrunculin B (Sigma-Aldrich) at different concentrations (0–50 µM) for 20 min at 37°C before washing in culture medium. Nalm6-treated cells were cocultured on MS5.1 cells for 2 h at 37°C, fixed and stained with appropriate Abs.

Latex beads (3 x 105) were used alone or loaded with 5 µg/ml anti-{alpha}5, -beta1, and -CD9 mAbs, saturated with 5% BSA and washed in PBS. Nalm6 cells (6 x 105) were preincubated for 15 min with hrGAL1 (10 µg/ml) on ice before incubation with the beads for 30 min at 37°C. The conjugates were allowed to settle on slides coated with poly-L-lysine, before being fixed and stained.

Flow cytometry

MS5.1 adherent cells (105 per test), were detached using trypsin-EDTA buffer (Invitrogen Life Technologies) and Nalm6 cells (2 x 105) were washed twice in PBA buffer before labeling for 45 min at 4°C using appropriate Abs. Analyses were performed using a FACSCalibur apparatus (BD Biosciences) and analyzed using CellQuest software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Integrins are galectin-1 counterreceptors on stromal and pre-B cell lines

We previously demonstrated that pre-BCR binding to the stromal cell-derived GAL1 lectin implicates direct protein/protein interactions. However, pre-BCR relocalization in the synapse formed between pre-B and stromal cells requires GAL1 binding to glycosylated counterreceptors expressed at the cell surface of stromal and pre-B cells (14).

To identify these counterreceptors, we first analyzed by Far Western blot the GAL1 binding pattern to stromal cell proteins. hrGAL1 binds to only a few murine and human stromal cell glycoproteins with m.w. comprised between 90 and 150 kDa (Fig. 1A). To identify these proteins, MS5.1 stromal cell lysate (3 x 108 cells) was incubated with 4 mg of hrGAL1-M2-His loaded onto nickel-Sepharose beads. Urea-eluted fractions were analyzed on a SDS 7.5–17.5% gradient PAGE. A differentially detected band at 130 kDa was excised from the preparative gel (data not shown) and analyzed by mass spectrometry. Peptide mass fingerprint revealed two proteins that were characterized as the {alpha}5 and beta1 integrins (SwissProt, P09055 and P11688, respectively). We confirmed by flow cytometry that {alpha}5 and beta1 integrins are present at the MS5.1 stromal cell surface (Fig. 1B) and that {alpha}5 and GAL1 can be coimmunoprecipitated from MS5.1 cell lysate using anti-beta1 mAb (Fig. 1C). These data demonstrated that {alpha}5beta1 integrins are GAL1 counterreceptors on the MS5.1 stromal cell line.


Figure 1
View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 1. Identification of integrins as GAL1 counterreceptors present on stromal and pre-B cells. A, Far Western blot analysis of Nalm6, MS5.1, and C8 cell line lysates was performed using hrGAL1 (2.5 µg/ml) detected by the rabbit anti-GAL1 AS and revealed by HRP-coupled protein A. B, FACS analysis of MS5.1 cell line using hamster anti-{alpha}5 and -beta1 mAbs revealed by FITC-goat anti-hamster Abs. C, MS5.1 stromal cell lysate was immunoprecipitated using hamster anti-beta1 and anti-CD3{epsilon} isotypic control (Ct) mAbs. Western blotting were performed using rabbit anti-{alpha}5 and -beta1 Abs and rabbit anti-GAL1 AS, revealed with HRP-coupled protein A. D, FACS analysis of Nalm6 cells using rabbit anti-{alpha}4, - {alpha}5, and -beta1 -beta7 Abs revealed by FITC-goat anti-rabbit Abs, mouse anti-human CD9 mAb revealed by FITC-goat anti-mouse Abs. E, Nalm6 (20 x 106 cells/test) were incubated with 10 µg/ml hrGAL1 in presence of 0.2 M lactose (L) or 0.2 M maltose (M). Left, Immunoprecipitations were performed using mouse anti-CD9 and -beta1 mAbs. In Western blots, beta1 integrin and CD9 were revealed using the same Abs, {alpha}4 and {alpha}5 integrins using rabbit anti-{alpha}4 and -{alpha}5 Abs and GAL1 using the rabbit anti-GAL1 AS. Rabbit and mouse Abs were revealed by HRP-protein A and by HRP-goat anti-mouse Abs, respectively. Right, Immunoprecipitations were performed using rabbit anti-{alpha}4, -{alpha}5, -beta1, -beta7 Abs and anti-CD22 mAb. Integrins and CD22 were revealed in Western blots using the same Abs and GAL1 using the rabbit anti-GAL1 AS. Rabbit and mouse Abs were revealed by HRP-protein A and by HRP-goat anti-mouse Abs, respectively.

 
As hrGAL1 revealed also molecules between 90 and 130 kDa on Nalm6 cell lysate in Far Western experiments (Fig. 1A), we have checked whether integrins could also be GAL1 counterreceptors on pre-B cells. Flow cytometry analysis showed that the Nalm6 pre-B cell line expresses {alpha}4, {alpha}5, beta1, and beta7 integrins at the cell surface (Fig. 1D). When Nalm6 pre-B cells were pretreated with hrGAL1, mAbs (mAb) against beta1 integrin coimmunoprecipitated GAL1 and also the {alpha}4 and {alpha}5 integrin chains. The interactions between integrins and GAL1 were lost upon lactose (a GAL1-specific sugar) and not maltose treatments, indicating that GAL1 binds to integrin glycans (Fig. 1E, left). hrGAL1 was also coimmunoprecipitated with {alpha}4, {alpha}5, beta1, and beta7 integrins, in a lactose sensible manner (Fig. 1E, right). We observe that anti-{alpha}4, {alpha}5, beta1, or beta7 Abs immunoprecipitate only the corresponding integrin chain. These results show that GAL1 binds directly to the {alpha}4, {alpha}5, beta1, and beta7 chains, however, the respective proportion of the different GAL1-integrin complexes cannot be determined from these nonquantitative experiments. As controls, hrGAL1 was not immunoprecipitated using a mAb against CD9, a member of the tetraspan superfamily that plays a role in cell adhesion and migration, nor using Abs against CD22, a negative regulator of B cell signaling, which is also heavily glycosylated (Fig. 1E).

These results show specific interactions between GAL1 and glycosylated integrins present on both pre-B and stromal cells, and are in agreement with previous data showing that integrins contain N-glycans recognized by galectins (20, 21, 22).

Pre-BCR, integrins, and their ligands form a homogeneous pre-B/stromal cell synapse

We next investigated whether the identified integrins are enriched in the developmental synapse and could therefore play a role in the pre-BCR relocalization into the pre-B/stromal cell synapse.

Confocal microscopy analysis of human Nalm6/MS5.1 conjugates performed using anti-{alpha}4, -{alpha}5, -beta1, or -beta7 Abs, revealed that integrin staining colocalizes with those of the pre-BCR and GAL1 molecules (Fig. 2). Because {alpha}4 and beta7 integrins are not expressed by the mouse MS5.1 stromal cell line (data not shown) and because the anti-beta1 mAb used is specific for the human beta1 integrin, we deduce that the {alpha}4, beta7, and beta1 integrins observed in the synapse are from pre-B cell origin. However, because anti-{alpha}5 Abs recognize both human and mouse {alpha}5 integrins, we cannot exclude that {alpha}5 integrins from stromal cell origin are also present into the synapse. CD9 and CD22 molecules that do not bind to GAL1 presented a different behavior: CD9 was never recruited at the contact zone between pre-B and stromal cells whereas CD22 was polarized but excluded from the pre-BCR area.


Figure 2
View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 2. Integrins, together with the pre-BCR and GAL1, relocalize into the synapse formed between pre-B and stromal cells. Nalm6 cells were cocultivated with MS5.1 stromal cells for 2 h and analyzed by confocal microscopy. Differential interference contrast images show fixed lymphoid/MS5.1 stromal cell conjugates. Staining was performed with: biotinylated goat anti-human µ Abs revealed by streptavidin-FITC or by streptavidin-rhodamine, mouse anti-VpreB mAb revealed by goat anti-mouse Texas Red Abs, hamster anti-{alpha}5 Abs revealed by FITC-goat anti-hamster Abs, rabbit anti-{alpha}4 and -beta7 Abs revealed by FITC-goat anti-rabbit Abs, goat anti-beta1 Abs revealed by Alexa 488-donkey anti-goat, mouse anti-beta1 and -CD9 mAbs revealed by Cya-5-goat anti-mouse Abs, rabbit anti-GAL1 AS revealed either by rhodamine-goat anti-rabbit or by Cya-5-goat anti-rabbit Abs and FITC-anti-CD22 mAb; m, merge view. Images are representative of three separate experiments. The percentages and the SDs of integrins and pre-BCR relocalization are indicated in Fig. 5.

 
To identify integrin ligands present on pre-B and stromal cells, we used confocal microscopy and flow cytometry. The fibronectin, a major component of the ECM, is a ligand for VLA-4, VLA-5, and {alpha}4beta7 integrins (23) and a potential GAL1 counterreceptor (24). By confocal microscopy, we observed that MS5.1 cells were able to produce a network of conventional fibronectin fibers (data not shown) and double staining revealed that fibronectin is clearly found at the pre-B/stromal cell interface where the pre-BCR is relocalized (Fig. 3A, bottom). Again, the repartition of CD9 molecules on pre-B cells was not modified after formation of cell/cell conjugates (Fig. 3A, top). ADAM15 and L1 are known as potential VLA-5 ligands (25) and the VCAM-1 is a VLA-4 and {alpha}4beta7 ligand (23). Flow cytometry experiments showed that the Nalm6 cell line is negative for both L1 and ADAM15 expression (Fig. 3B) and that the MS5.1 cell line is negative for L1 but positive for VCAM1 and ADAM15 (Fig. 3C). The lack of L1 expression on these cells was confirmed using two distinct anti-L1 mAbs (data not shown). VCAM1 staining was too weak to observe any accumulation at the stromal/pre-B interface (data not shown). In contrast, the ADAM15/pre-BCR double staining showed that the two molecules colocalize and are polarized at the contact zone between the two cells (Fig. 3D).


Figure 3
View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 3. Fibronectin and ADAM-15 integrin ligands are found in the pre-B/stromal cell synapse. A, Double staining of Nalm6 cells cultivated on MS5.1 stromal cells during 2 h, using rabbit anti-fibronectin Abs revealed by rhodamine-goat anti-rabbit Abs and mouse anti-CD9 and -VpreB mAbs revealed by FITC-goat anti-mouse Abs are analyzed by confocal microscopy. B and C, FACS analysis on the Nalm6 and the MS5.1 cell lines, respectively, using rat anti-L1 mAb revealed by FITC-goat anti-rat Abs, goat anti-ADAM-15 Abs revealed by Alexa 488-donkey anti-goat Abs, and by FITC-rat anti-VCAM1 mAbs. D, Double staining of Nalm6 cells cultivated on MS5.1 stromal cells, using goat anti-ADAM-15 Abs and the anti-VpreB mAb, revealed by Alexa 488-donkey anti-goat and Texas Red-goat anti-mouse Abs, respectively. DIC images show fixed lymphoid/MS5.1 stromal cell conjugates; m, merge view. Images are representative of three separate experiments.

 
These data indicate that the VLA-4 and VLA-5 integrins on pre-B cells and the ADAM15 integrin ligand on stromal cells accumulate with the pre-BCR and GAL1 at the contact area between pre-B and stromal cells. Thus, as it was described for T and NK cells (26, 27), adhesion molecules seem to play a major role in the organization of the pre-B/stromal cell synapse. In immunological synapses, constituted of a central supramolecular activation cluster (cSMAC) and a peripheral SMAC (pSMAC), integrins are usually found segregated into the pSMAC (26, 27). Interestingly, during pre-B/stromal synapse formation, we never observed formation of cSMAC or pSMAC. Instead, pre-BCR, integrins and their ligands always colocalized, forming a homogeneous platform, as illustrated by confocal Z sectioning (Fig. 4 and data not shown).


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. Pre-BCR, galectin-1, and integrins form a homogeneous synapse. Nalm6 cells were cocultivated with MS5.1 stromal cells for 2 h and analyzed by confocal microscopy. Staining was performed with biotinylated goat anti-human µ Abs revealed by streptavidin-rhodamine and anti-{alpha}5 mAb revealed by Cya-5-goat anti-mouse Abs. One representative confocal section, the DIC image, and the combined xy, xz, and yz view are represented.

 
Integrin and pre-BCR relocalization is synchronized and requires actin polymerization

As integrin interactions with their ligands are involved in integrin polarization (28), we hypothesized that this phenomenon could play a role in the pre-BCR relocalization process.

The pre-BCR relocalization has been previously monitored at 5 min, 30 min, and 2 h of pre-B/stromal cells cocultures (14). Using the same technique, we observed that the relocalization time courses of the different integrins and the pre-BCR were very similar, whatever the time of cocultures (Fig. 5A), suggesting that movements of pre-BCR and integrins are synchronized. A representative observation field of pre-B cells, presenting relocalized {alpha}5 integrins after 2 h of coculture with stromal cells, is depicted in Fig. 5B. As integrin mobility depends on actin cytoskeleton, we tested the implication of actin polymerization on integrin and pre-BCR relocalization. Pre-B cells were incubated with latrinculin B, an inhibitor of actin polymerization, before a 2-h coculture with the MS5.1 stromal cells. We observed that this treatment did not abrogate the adhesion of pre-B cells to stromal cells, but the percentage of cells with a relocalized pre-BCR diminished in a dose-dependent manner, with a maximum inhibition of 70% in presence of 5 µM latrunculin B (data not shown). At this latrunculin B concentration, relocalization of the different integrins is efficiently inhibited and is comparable to that of the pre-BCR (Fig. 5C). These data suggest that the processes of integrin and pre-BCR relocalization are linked and require the reorganization of actin cytoskeleton in pre-B cells.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5. Analysis of integrin and pre-BCR relocalization. A, Percentage of cells with relocalized {alpha}4, {alpha}5, beta1, and pre-BCR at 5 min, 30 min, and 2 h of Nalm6/MS5.1 cocultures. Cells were counted after staining and confocal microscopy analysis. Rabbit anti-{alpha}4, -{alpha}5, and -beta1 Abs were revealed using Rhodamine-goat anti-rabbit Abs. The pre-BCR was stained using the anti-VpreB mAb revealed by Texas Red-goat anti-mouse Abs. B, A representative view of pre-B/stromal cell conjugates after 2-h coculture, labeled with anti-{alpha}5 Abs and revealed by goat anti-rabbit Abs coupled to FITC. Enlarged views 1 and 2 show relocalized and nonrelocalized cells, respectively. C, Percentage of cells with relocalized {alpha}4, {alpha}5, beta1, beta7, and pre-BCR when Nalm6 cells were incubated with 5 µM latrunculin B before 2-h coculture with MS5.1 cells. For beta7 detection, rabbit anti-beta7 Abs were used and revealed using rhodamine-goat anti-rabbit Abs. D, Percentage of cells with relocalized {alpha}4, {alpha}5, and pre-BCR after Nalm6/MS5.1 cocultures in presence of 0.2 M maltose or 0.2 M lactose, during 2 h. A–D, Each experiment was performed in duplicate and 130–320 cells were counted using x16 magnification images.

 
Pre-B cell integrin cross-linking leads to GAL1-dependent pre-BCR clustering and activation

When pre-B and stromal cells cocultures were performed in the presence of lactose, the pre-BCR relocalization process was inhibited (Fig. 5D and (14)) whereas that of {alpha}4 and {alpha}5 integrins was not altered (Fig. 5D). These results show that GAL1/integrin interactions are not necessary for integrin clustering and suggest that the active relocalization of integrins at the stromal/pre-B cells interface is implicated in driving the pre-BCR into the synapse. To test this hypothesis in a simple way, we decided to mimic the role of stromal integrin ligand(s) in the clustering of pre-B cell integrins. To that, we incubated latex beads coupled with anti-{alpha}5 and beta1 integrins mAbs with Nalm6 cells in presence or absence of hrGAL1 and we followed the pre-BCR relocalization (Fig. 6A). In these conditions, we observed that the pre-BCR staining was recruited at the contact cells/beads area only in presence of GAL1. In contrast, when pre-B cells interact with latex beads alone or with beads coupled with anti-CD9 mAb in presence of GAL1, the pre-BCR was not enriched at the interacting zone. These results demonstrate that GAL1 creates a link between the pre-BCR and the relocalizing integrins.


Figure 6
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 6. Pre-B cell integrins drive pre-BCR relocalization and activation. A, Nalm6 cells were cultivated 30 min at 37°C with latex beads alone or coupled with anti-beta1, -{alpha}5, and -CD9 mAbs, in the absence (left) or presence (right) of hrGAL1. Staining was performed with biotinylated goat anti-human µ Abs revealed by streptavidin-Alexa 488 and analyzed by confocal microscopy. DIC images show fixed cell/latex bead conjugates. Few cell conjugates were formed when beads alone were used. For beads coupled with mAbs, 150 conjugates were analyzed in two separate experiments. Using beads coated with anti-integrins and anti-CD9 mAbs in the presence of hrGAL1, 32 ± 7.0% and 5.5 ± 2.5% of pre-BCR were relocalized, respectively. B, Nalm6 cells were cultivated 30 min at 37°C with latex beads coupled with anti-beta1 and -CD9 mAbs, in the absence or presence of hrGAL1. Then, conjugates were lysed and immunoprecipitations were performed using mouse anti-CD79a mAb. Phosphorylated proteins were revealed using the 4G10 mAb, CD79a protein using the same mAb as for immunoprecipitations and Lyn protein using rabbit anti-Lyn Abs. Rabbit and mouse Abs were revealed using HRP-protein-A and HRP-goat anti-mouse Abs, respectively.

 
To test the impact of integrin cross-linking on pre-BCR signaling, we analyzed CD79a phosphorylation and its association to Lyn, using the above latex bead experimental system. Pre-B cell-latex bead conjugates were harvested after 30-min incubation, lysed, and CD79a was immunoprecipitated. After Western blotting using the 4G10 anti-phosphotyrosine mAb, we observed that in the presence of GAL1, beta1 integrin cross-linking induced CD79a and Lyn phosphorylation (Fig. 6B, left). The phosphorylated bands at 42 and 55 kDa correspond to CD79a and to the upper form of the Lyn protein doublet, respectively, as revealed by Western blotting using anti-CD79a and anti-Lyn Abs. We cannot determine the phosphorylation status of the lower form of Lyn because it comigrates with the immunoprecipitating Ab H chain. We also observed that beta1 cross-linking enhances Lyn recruitment to CD79a, only when GAL1 is present. By contrast, CD9 cross-linking has no effect either on Lyn recruitment or Lyn and CD79a phosphorylation, even in the presence of GAL1 (Fig. 6B).

Altogether, these data demonstrate that pre-B cell integrin cross-linking is sufficient to cluster pre-BCRs and to initiate pre-BCR signaling in the presence of GAL1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We previously showed that pre-BCR translocation into the pre-B/stromal cell synapse initiates intracellular tyrosine kinase activity and signal transduction from the pre-BCR. During this process, pre-BCRs bind directly to GAL1 molecules, which are anchored to glycosylated cell surface receptors. However, the nature and the cell origin of these receptors, either from stromal and/or pre-B cells, were not determined (14). In this study, we show that integrins expressed by pre-B cells are the major GAL1 counterreceptors implicated in synapse formation, pre-BCR clustering, and pre-BCR activation.

We show that GAL1 binds to VLA-5 integrins on stromal cells and to VLA-4, VLA-5, and {alpha}4beta7 integrins on pre-B cells. Moreover, we demonstrate that integrins and GAL1 are part of the synapse formed between pre-B and stromal cells. Integrins are present in their active conformation because they are stained by the HUTS anti-beta1 mAb, which detects the activated conformation of the beta1 integrin (29) (data not shown). GAL1 has been already shown to bind to a variety of molecules including members of the integrin family, such as {alpha}7beta1 (30), beta1 integrin (31) but also to ECM components, such as laminin and fibronectin (24, 30). Moreover, it was demonstrated that GAL1 could increase cell adhesion, through beta1 integrin activation (31). Finally, among members of the galectin family, GAL3 and GAL8 were also found to interact with integrins (32, 33). We identify ADAM15, fibronectin, and possibly VCAM1 as integrin ligands expressed by stromal cells and which are present at the intercellular junction between pre-B and stromal cells. As integrin-ligand pairs are already known (23), we postulate that VLA-5 on pre-B cells interact with ADAM15 and/or fibronectin on stromal cells and that VLA-4 and {alpha}4beta7 on pre-B cells interact with fibronectin and/or VCAM1 on stromal cells. Altogether, our data indicate that GAL1 is part of an adhesion platform between pre-B and stromal cells, formed by integrins and their ligands.

For NK and T cells, LFA-1/ICAM-1 adhesion is the first stage of synapse formation that is followed by perforin or TCR migration within cSMAC and by integrins repositioning within the pSMAC (26, 27). VLA-4 integrin also concentrates at the pSMAC in mature T cells (34). In contrast, for immature CD4+CD8+ thymocytes, LFA-1 and TCR molecules are organized in a multifocal immunological synapse (35). In the case of a mature B synapse, an LFA-1 ring is observed around the BCR (36), the CD45 coreceptor is excluded, and the CD22-negative regulator is depleted from the synapse (28, 37). During pre-B/stromal synapse formation, the pre-BCR and the integrins are always observed colocalized and form a homogeneous lattice (Fig. 4 and data not shown). For T cell synapses, it was proposed that exclusion of long molecules, as integrins, into the pSMAC is a prerequisite to promote stable TCR/MHC contacts (38). In the case of the pre-B/stromal cell synapse, such a segregation mechanism is not observed because the pre-BCR does not bind directly to a stromal cell transmembrane receptor, but to secreted GAL1 anchored to pre-B integrin counterreceptors.

Integrin-ligand interactions participate in the adhesion of B cell progenitors with the BM environment and are critical for sustaining B lymphopoiesis (39, 40). VLA-4 and VLA-5 are particularly important to promote both pro-B cell adhesion and proliferation (41, 42). Moreover, {alpha}4 in association with beta1 or beta7 integrins, plays an important role in maintaining normal hemopoiesis (5). ECM components also provide a network in which different soluble factors secreted by stromal cells, such as CXCL12 and IL-7, can be trapped. CXCL12 sequestration into the fibronectin lattice is implicated in CXCR4 redistribution at the cell surface of early B cell progenitors (43) and activates LFA-1, VLA-4, and VLA-5 integrins (44). ADAM-15 is a metalloprotease implicated in the degradation of the ECM (45) and its accumulation in the synapse may promote pre-B cell mobility on the stromal cell surface. These observations lead to the conclusion that integrins and their ligands, but also chemokines and their receptors, could participate in the polarization process leading to an organized intercellular junction between pre-B and stromal cells. In our system, we demonstrate that synapse formation is dependent on actin polymerization because integrins and pre-BCR relocalization are lost when pre-B cells are treated with latrunculin B. Examples of galectin-glycoprotein lattices have already been described (46, 47) suggesting that GAL1 could participate in the pre-B/stromal cell synapse by the formation of an organized lattice between integrins, the ECM and the pre-BCR. We reported that lactose treatment does not abrogate pre-B/stromal cell adhesion and does not abolish integrin relocalization, leading to the hypothesis that GAL1 forms a molecular link between the pre-BCR and the relocalizing integrins. Indeed, the induction of pre-B cell integrin clustering using anti-integrin mAbs-coated beads induces pre-BCR relocalization only in presence of GAL1 (Fig. 6A). Moreover, cross-linking of pre-B cell integrins is sufficient to initiate pre-BCR signaling in the presence of GAL1, leading to Lyn recruitment and to Lyn and CD79a phosphorylation.

Therefore, we propose that formation of the pre-B/stromal cell synapse proceeds in two steps (Fig. 7): 1) the stromal cell-derived GAL1 binds to pre-B cell glycosylated integrins and directly to the pre-BCR, and 2) the active pre-B cell integrin relocalization, mediated by interactions with their stromal cell ligands, drives pre-BCR relocalization into the synapse and initiates pre-BCR signaling. In the synapse, CD22 exclusion from the pre-BCR area also emphasizes the fact that pre-BCR clustering leads to positive cell signaling. The lattice generated by integrin-GAL1-pre-BCR interactions may regroup the different pre-BCR into the synapse to reach the activation threshold necessary to initiate pre-BCR differentiation/proliferation programs and also to reinforce the adhesion of B cell precursors to stromal cells.


Figure 7
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 7. Molecular organization of the pre-B/stromal cell synapse. A model of the molecular organization of the synapse: GAL1 secreted by stromal cells is captured by relocalizing pre-B cell integrins interacting with their ligands on stromal cells and by pre-BCR. The active integrin relocalization in the presence of GAL1 drives the pre-BCR into the pre-B/stromal cell synapse, leading to formation of a homogeneous lattice and to initiation of pre-BCR signaling.

 

   Acknowledgments
 
We thank M. Fallet for excellent assistance with confocal microscopy. We gratefully acknowledge M. Court and J. Garin from the Laboratoire de Chimie des Protéines, Commissariat à l’Energie Atomique, Grenoble, France, for mass spectrometry analysis. We are grateful to Drs. A. C. Lellouch, S. Salcedo, and H. Lelouard for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by CNRS, INSERM, Agence Nationale de la Recherche (ANR) (NT05-341681), and Association pour la Recherche contre le Cancer (ARC), Contracts Nos. 4808 and 3656. B.R. was funded by the INSERM-Region PACA and by ARC. Back

2 Address correspondence and reprint requests to Dr. Claudine Schiff, CIML, Case 906, 13288 Marseille Cedex 09, France. E-mail address: schiff{at}ciml.univ-mrs.fr Back

3 Current address: Innate-Pharma, 119/121 chemin de Cassis, 13009 Marseille, France. Back

4 Abbreviations used in this paper: BM, bone marrow; SLC, surrogate L chain; ECM, extracellular matrix; GAL1, galectin-1; AS, antiserum; hr, human recombinant; SMAC, supramolecular activation cluster; cSMAC, central SMAC; pSMAC, peripheral SMAC. DIC, different interference contrast; CXCL12, stromal cell-derived factor-1. Back

Received for publication September 9, 2005. Accepted for publication April 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hardy, R. R., K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Immunol. 19: 595-621. [Medline]
  2. Melchers, F., E. ten Boekel, T. Seidl, X. C. Kong, T. Yamagami, K. Onishi, T. Shimizu, A. G. Rolink, J. Andersson. 2000. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175: 33-46. [Medline]
  3. LeBien, T. W.. 1998. B-cell lymphopoiesis in mouse and man. Curr. Opin. Immunol. 10: 188-195. [Medline]
  4. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11-25. [Medline]
  5. Arroyo, A. G., J. T. Yang, H. Rayburn, R. O. Hynes. 1999. {alpha}4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 11: 555-566. [Medline]
  6. Egawa, T., K. Kawabata, H. Kawamoto, K. Amada, R. Okamoto, N. Fujii, T. Kishimoto, Y. Katsura, T. Nagasawa. 2001. The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor. Immunity 15: 323-334. [Medline]
  7. Lyman, S. D., S. E. Jacobsen. 1998. c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 91: 1101-1134. [Free Full Text]
  8. Sudo, T., M. Ito, Y. Ogawa, M. Iizuka, H. Kodama, T. Kunisada, S. Hayashi, M. Ogawa, K. Sakai, S. Nishikawa. 1989. Interleukin 7 production and function in stromal cell-dependent B cell development. J. Exp. Med. 170: 333-338. [Abstract/Free Full Text]
  9. Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, K. Rajewsky. 1992. A critical role of {lambda}5 protein in B cell development. Cell 69: 823-831. [Medline]
  10. Kitamura, D., K. Rajewsky. 1992. Targeted disruption of µ chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356: 154-156. [Medline]
  11. Loffert, D., A. Ehlich, W. Muller, K. Rajewsky. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4: 133-144. [Medline]
  12. Keyna, U., G. B. Beck-Engeser, J. Jongstra, S. E. Applequist, H. M. Jack. 1995. Surrogate light chain-dependent selection of Ig heavy chain V regions. J. Immunol. 155: 5536-5542. [Abstract]
  13. ten Boekel, E., F. Melchers, A. G. Rolink. 1997. Changes in the VH gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7: 357-368. [Medline]
  14. Gauthier, L., B. Rossi, F. Roux, E. Termine, C. Schiff. 2002. Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering. Proc. Natl. Acad. Sci. USA 99: 13014-13019. [Abstract/Free Full Text]
  15. Bradl, H., J. Wittmann, D. Milius, C. Vettermann, H. M. Jack. 2003. Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of {lambda}5 and stroma cell-associated heparan sulfate. J. Immunol. 171: 2338-2348. [Abstract/Free Full Text]
  16. Ohnishi, K., F. Melchers. 2003. The nonimmunoglobulin portion of {lambda}5 mediates cell-autonomous pre-B cell receptor signaling. Nat. Immunol. 4: 849-856. [Medline]
  17. Guo, B., R. M. Kato, M. Garcia-Lloret, M. I. Wahl, D. J. Rawlings. 2000. Engagement of the human pre-B cell receptor generates a lipid raft- dependent calcium signaling complex. Immunity 13: 243-253. [Medline]
  18. Lemmers, B., L. Gauthier, V. Guelpa-Fonlupt, M. Fougereau, C. Schiff. 1999. The human (PsiL+µ–) proB complex: cell surface expression and biochemical structure of a putative transducing receptor. Blood 93: 4336-4346. [Abstract/Free Full Text]
  19. Castellani, V., E. De Angelis, S. Kenwrick, G. Rougon. 2002. Cis and trans interactions of L1 with neuropilin-1 control axonal responses to semaphorin 3A. EMBO J. 21: 6348-6357. [Medline]
  20. Nakayama, J., J. C. Yeh, A. K. Misra, S. Ito, T. Katsuyama, M. Fukuda. 1999. Expression cloning of a human {alpha}1, 4-N-acetylglucosaminyltransferase that forms GlcNAc{alpha}1->4Galbeta->R, a glycan specifically expressed in the gastric gland mucous cell-type mucin. Proc. Natl. Acad. Sci. USA 96: 8991-8996. [Abstract/Free Full Text]
  21. Asada, M., K. Furukawa, C. Kantor, C. G. Gahmberg, A. Kobata. 1991. Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18. Biochemistry 30: 1561-1571. [Medline]
  22. Fischer, C., H. Sanchez-Ruderisch, M. Welzel, B. Wiedenmann, T. Sakai, S. Andre, H. J. Gabius, L. Khachigian, K. M. Detjen, S. Rosewicz. 2005. Galectin-1 interacts with the {alpha}5beta1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27. J. Biol. Chem. 280: 37266-37277. [Abstract/Free Full Text]
  23. van der Flier, A., A. Sonnenberg. 2001. Function and interactions of integrins. Cell Tissue Res. 305: 285-298. [Medline]
  24. Ozeki, Y., T. Matsui, Y. Yamamoto, M. Funahashi, J. Hamako, K. Titani. 1995. Tissue fibronectin is an endogenous ligand for galectin-1. Glycobiology 5: 255-261. [Abstract/Free Full Text]
  25. Nath, D., P. M. Slocombe, P. E. Stephens, A. Warn, G. R. Hutchinson, K. M. Yamada, A. J. Docherty, G. Murphy. 1999. Interaction of metargidin (ADAM-15) with {alpha}vbeta3 and {alpha}5beta1 integrins on different haemopoietic cells. J. Cell Sci. 112: 579-587. [Abstract]
  26. Orange, J. S., K. E. Harris, M. M. Andzelm, M. M. Valter, R. S. Geha, J. L. Strominger. 2003. The mature activating natural killer cell immunologic synapse is formed in distinct stages. Proc. Natl. Acad. Sci. USA 100: 14151-14156. [Abstract/Free Full Text]
  27. Lee, K. H., A. R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma, T. N. Sims, W. R. Burack, H. Wu, J. Wang, et al 2003. The immunological synapse balances T cell receptor signaling and degradation. Science 302: 1218-1222. [Abstract/Free Full Text]
  28. Carrasco, Y. R., F. D. Batista. 2006. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. EMBO J. 25: 889-899. [Medline]
  29. Luque, A., M. Gomez, W. Puzon, Y. Takada, F. Sanchez-Madrid, C. Cabanas. 1996. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common beta1 chain. J. Biol. Chem. 271: 11067-11075. [Abstract/Free Full Text]
  30. Gu, M., W. Wang, W. K. Song, D. N. Cooper, S. J. Kaufman. 1994. Selective modulation of the interaction of {alpha}7beta1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation. J. Cell Sci. 107: (Pt. 1):175-181. [Abstract]
  31. Moiseeva, E. P., B. Williams, A. H. Goodall, N. J. Samani. 2003. Galectin-1 interacts with beta-1 subunit of integrin. Biochem. Biophys. Res. Commun. 310: 1010-1016. [Medline]
  32. Fukumori, T., Y. Takenaka, T. Yoshii, H. R. Kim, V. Hogan, H. Inohara, S. Kagawa, A. Raz. 2003. CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis. Cancer Res. 63: 8302-8311. [Abstract/Free Full Text]
  33. Hadari, Y. R., R. Arbel-Goren, Y. Levy, A. Amsterdam, R. Alon, R. Zakut, Y. Zick. 2000. Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis. J. Cell Sci. 113: 2385-2397. [Abstract]
  34. Mittelbrunn, M., A. Molina, M. M. Escribese, M. Yanez-Mo, E. Escudero, A. Ursa, R. Tejedor, F. Mampaso, F. Sanchez-Madrid. 2004. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses. Proc. Natl. Acad. Sci. USA 101: 11058-11063. [Abstract/Free Full Text]
  35. Hailman, E., W. R. Burack, A. S. Shaw, M. L. Dustin, P. M. Allen. 2002. Immature CD4+CD8+ thymocytes form a multifocal immunological synapse with sustained tyrosine phosphorylation. Immunity 16: 839-848. [Medline]
  36. Carrasco, Y. R., S. J. Fleire, T. Cameron, M. L. Dustin, F. D. Batista. 2004. LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20: 589-599. [Medline]
  37. Batista, F. D., D. Iber, M. S. Neuberger. 2001. B cells acquire antigen from target cells after synapse formation. Nature 411: 489-494. [Medline]
  38. Shaw, A. S., M. L. Dustin. 1997. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 6: 361-369. [Medline]
  39. McGinnes, K., V. Quesniaux, J. Hitzler, C. Paige. 1991. Human B-lymphopoiesis is supported by bone marrow-derived stromal cells. Exp. Hematol. 19: 294-303. [Medline]
  40. Jacobsen, K., D. G. Osmond. 1990. Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow. Eur. J. Immunol. 20: 2395-2404. [Medline]
  41. Qiang, Y. W., M. Kitagawa, M. Higashi, G. Ishii, C. Morimoto, K. Harigaya. 2000. Activation of mitogen-activated protein kinase through {alpha}5/beta1 integrin is required for cell cycle progression of B progenitor cell line, Reh, on human marrow stromal cells. Exp. Hematol. 28: 1147-1157. [Medline]
  42. Hahn, B. K., D. Piktel, L. F. Gibson, K. S. Landreth. 2000. Hematopoiesis: the role of stromal integrin interactions in pro-B cell proliferation. Hematology 5: 153-160. [Medline]
  43. van Buul, J. D., C. Voermans, J. van Gelderen, E. C. Anthony, C. E. van der Schoot, P. L. Hordijk. 2003. Leukocyte-endothelium interaction promotes SDF-1-dependent polarization of CXCR4. J. Biol. Chem. 278: 30302-30310. [Abstract/Free Full Text]
  44. Peled, A., O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, M. M. Slav, A. Nagler, O. Lider, R. Alon, et al 2000. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95: 3289-3296. [Abstract/Free Full Text]
  45. Martin, J., L. V. Eynstone, M. Davies, J. D. Williams, R. Steadman. 2002. The role of ADAM 15 in glomerular mesangial cell migration. J. Biol. Chem. 277: 33683-33689. [Abstract/Free Full Text]
  46. Pace, K. E., C. Lee, P. L. Stewart, L. G. Baum. 1999. Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1. J. Immunol. 163: 3801-3811. [Abstract/Free Full Text]
  47. Demetriou, M., M. Granovsky, S. Quaggin, J. W. Dennis. 2001. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature 409: 733-739. [Medline]



This article has been cited by other articles:


Home page
 Protein Sci.Home page
L. Morstadt, A. Bohm, D. Yuksel, K. Kumar, B. D. Stollar, and J. D. Baleja
Engineering and characterization of a single chain surrogate light chain variable domain
Protein Sci., March 1, 2008; 17(3): 458 - 465.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
W. Li, K. Ishihara, T. Yokota, T. Nakagawa, N. Koyama, J. Jin, Y. Mizuno-Horikawa, X. Wang, E. Miyoshi, N. Taniguchi, et al.
Reduced {alpha}4 1 Integrin/VCAM-1 Interactions Lead to Impaired Pre-B Cell Repopulation in Alpha 1,6-Fucosyltransferase Deficient Mice
Glycobiology, January 1, 2008; 18(1): 114 - 124.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. A. Martin, L. Lu, M. Cascalho, and G. E. Wu
Maintenance of Surrogate Light Chain Expression Induces Developmental Delay in Early B Cell Compartment
J. Immunol., October 15, 2007; 179(8): 4996 - 5005.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rossi, B.
Right arrow Articles by Gauthier, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rossi, B.
Right arrow Articles by Gauthier, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS